Steroids in human serum are bound to proteins. In clinical chemical hormone analysis, hormone determinations are based almost entirely on the principle of dissolving the bond between the transport hormones and the hormones and to measure the total concentration of the hormone. The techniques used to dissolve the bonds may be of the most varied types, such as, solvent extraction, heat denaturization, enzymatic hydrolysis and the treatment with acids or alkalis.
However, the content of bonding proteins in the human serum is not always of the same magnitude. It is known, for example, that female patients under antiovulation therapy or during late pregnancy exhibit clearly higher values of cortisol-binding globulin or thyroxine-binding globulin. Other examples include the isolated controversial increases or decreases in bonding protein contents found in different individuals of related familial background and heredity.
Bonding protein levels varying in this manner often result in the determination of total hormones to values which indicate a certain syndrome, while the patients do not show the clinical image of the corresponding syndrome. Apparently, this is due to the fact that the results reflect only the portion not bound to proteins, while the portion bound to the protein is biologically inactive.
The known techniques of the determination of protein-bound or free hormones are either those in which the equilibrium between the bound and free hormones is maintained during the separation, such as, for example, in classical equilibrium dialysis or ultrafiltration, or those in which the equilibrium is not maintained during the separation, such as, for example, column chromatography or adsorption methods. These techniques, nevertheless, yield a measure of the portion of the hormones that is actually free. In these techniques, the reaction time is a critical factor because the bond between the adsorbing agent and the steroid is irreversible.
Among the disadvantages of the known separation methods is that they are very expensive and yield only the percentual proportion of the free hormone. The absolute value must be calculated through the additional determination of total hormone concentration. Because of this indirect determination, measurements become correspondingly inaccurate, aside from the highly expensive techniques required to determine the percentual proportion.